Method for rapid detection and identification of bioagents

ABSTRACT

Method for detecting and identifying unknown bioagents, including bacteria, viruses and the like, by a combination of nucleic acid amplification and molecular weight determination using primers which hybridize to conserved sequence regions of nucleic acids derived from a bioagent and which bracket variable sequence regions that uniquely identify the bioagent. The result is a “base composition signature” (BCS) which is then matched against a database of base composition signatures, by which the bioagent is identified.

STATEMENT OF GOVERNMENT SUPPORT

[0001] This invention was made with U.S. Government support underDARPA/SPO contract BAA00-09. The U.S. Government may have certain rightsin the invention.

FIELD OF THE INVENTION

[0002] The present invention relates to methods for rapid detection andidentification of bioagents from environmental, clinical or othersamples. The methods provide for detection and characterization of aunique base composition signature (BCS) from any bioagent, includingbacteria and viruses. The unique BCS is used to rapidly identify thebioagent.

BACKGROUND OF THE INVENTION

[0003] Rapid and definitive microbial identification is desirable for avariety of industrial, medical, environmental, quality, and researchreasons. Traditionally, the microbiology laboratory has functioned toidentify the etiologic agents of infectious diseases through directexamination and culture of specimens. Since the mid-1980s, researchershave repeatedly demonstrated the practical utility of molecular biologytechniques, many of which form the basis of clinical diagnostic assays.Some of these techniques include nucleic acid hybridization analysis,restriction enzyme analysis, genetic sequence analysis, and separationand purification of nucleic acids (See, e.g., J. Sambrook, E. F.Fritsch, and T. Maniatis, Molecular Cloning: A Laboratory Manual, 2ndEd., Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y.,1989). These procedures, in general, are time-consuming and tedious.Another option is the polymerase chain reaction (PCR) or otheramplification procedure which amplifies a specific target DNA sequencebased on the flanking primers used. Finally, detection and data analysisconvert the hybridization event into an analytical result.

[0004] Other techniques for detection of bioagents includehigh-resolution mass spectrometry (MS), low-resolution MS, fluorescence,radioiodination, DNA chips and antibody techniques. None of thesetechniques is entirely satisfactory.

[0005] Mass spectrometry provides detailed information about themolecules being analyzed, including high mass accuracy. It is also aprocess that can be easily automated. However, high-resolution MS alonefails to perform against unknown or bioengineered agents, or inenvironments where there is a high background level of bioagents(“cluttered” background). Low-resolution MS can fail to detect someknown agents, if their spectral lines are sufficiently weak orsufficiently close to those from other living organisms in the sample.DNA chips with specific probes can only determine the presence orabsence of specifically anticipated organisms. Because there arehundreds of thousands of species of benign bacteria, some very similarin sequence to threat organisms, even arrays with 10,000 probes lack thebreadth needed to detect a particular organism.

[0006] Antibodies face more severe diversity limitations than arrays. Ifantibodies are designed against highly conserved targets to increasediversity, the false alarm problem will dominate, again because threatorganisms are very similar to benign ones. Antibodies are only capableof detecting known agents in relatively uncluttered environments.

[0007] Several groups have described detection of PCR products usinghigh resolution electrospray ionization-Fourier transform-ion cyclotronresonance mass spectrometry (ESI-FT-ICR MS). Accurate measurement ofexact mass combined with knowledge of the number of at least onenucleotide allowed calculation of the total base composition for PCRduplex products of approximately 100 base pairs. (Aaserud et al., J. Am.Soc. Mass Spec. 7:1266-1269, 1996; Muddiman et al., Anal. Chem.69:1543-1549, 1997; Wunschel et al., Anal. Chem. 70:1203-1207, 1998;Muddiman et al., Rev. Anal. Chem. 17:1-68, 1998). Electrosprayionization-Fourier transform-ion cyclotron resistance (ESI-FT-ICR) MSmay be used to determine the mass of double-stranded, 500 base-pair PCRproducts via the average molecular mass (Hurst et al., Rapid Commun.Mass Spec. 10:377-382, 1996). The use of matrix-assisted laserdesorption ionization-time of flight (MALDI-TOF) mass spectrometry forcharacterization of PCR products has been described. (Muddiman et al.,Rapid Commun. Mass Spec. 13:1201-1204, 1999). However, the degradationof DNAs over about 75 nucleotides observed with MALDI limited theutility of this method.

[0008] U.S. Pat. No. 5,849,492 describes a method for retrieval ofphylogenetically informative DNA sequences which comprise searching fora highly divergent segment of genomic DNA surrounded by two highlyconserved segments, designing the universal primers for PCRamplification of the highly divergent region, amplifying the genomic DNAby PCR technique using universal primers, and then sequencing the geneto determine the identity of the organism.

[0009] U.S. Pat. No. 5,965,363 discloses methods for screening nucleicacids for polymorphisms by analyzing amplified target nucleic acidsusing mass spectrometric techniques and to procedures for improving massresolution and mass accuracy of these methods.

[0010] WO 99/14375 describes methods, PCR primers and kits for use inanalyzing preselected DNA tandem nucleotide repeat alleles by massspectrometry.

[0011] WO 98/12355 discloses methods of determining the mass of a targetnucleic acid by mass spectrometric analysis, by cleaving the targetnucleic acid to reduce its length, making the target single-stranded andusing MS to determine the mass of the single-stranded shortened target.Also disclosed are methods of preparing a double-stranded target nucleicacid for MS analysis comprising amplification of the target nucleicacid, binding one of the strands to a solid support, releasing thesecond strand and then releasing the first strand which is then analyzedby MS. Kits for target nucleic acid preparation are also provided.

[0012] PCT WO97/33000 discloses methods for detecting mutations in atarget nucleic acid by nonrandomly fragmenting the target into a set ofsingle-stranded nonrandom length fragments and determining their massesby MS.

[0013] U.S. Pat. No. 5,605,798 describes a fast and highly accurate massspectrometer-based process for detecting the presence of a particularnucleic acid in a biological sample for diagnostic purposes.

[0014] WO 98/21066 describes processes for determining the sequence of aparticular target nucleic acid by mass spectrometry. Processes fordetecting a target nucleic acid present in a biological sample by PCRamplification and mass spectrometry detection are disclosed, as aremethods for detecting a target nucleic acid in a sample by amplifyingthe target with primers that contain restriction sites and tags,extending and cleaving the amplified nucleic acid, and detecting thepresence of extended product, wherein the presence of a DNA fragment ofa mass different from wild-type is indicative of a mutation. Methods ofsequencing a nucleic acid via mass spectrometry methods are alsodescribed.

[0015] WO 97/37041, WO 99/31278 and U.S. Pat. No. 5,547,835 describemethods of sequencing nucleic acids using mass spectrometry. U.S. Pat.Nos. 5,622,824, 5,872,003 and 5,691,141 describe methods, systems andkits for exonuclease-mediated mass spectrometric sequencing. Thus, thereis a need for a method for bioagent detection and identification whichis both specific and rapid, and in which no nucleic acid sequencing isrequired. The present invention addresses this need.

SUMMARY OF THE INVENTION

[0016] One embodiment of the present invention is a method ofidentifying an unknown bioagent comprising (a) contacting nucleic acidfrom the bioagent with at least one pair of oligonucleotide primerswhich hybridize to sequences of the nucleic acid and flank a variablenucleic acid sequence; (b) amplifying the variable nucleic acid sequenceto produce an amplification product; (c) determining the molecular massof the amplification product; and (d) comparing the molecular mass toone or more molecular masses of amplification products obtained byperforming steps (a)-(c) on a plurality of known organisms, wherein amatch identifies the unknown bioagent. In one aspect of this preferredembodiment, the sequences to which the at least one pair ofoligonucleotide primers hybridize are highly conserved. Preferably, theamplifying step comprises polymerase chain reaction. Alternatively, theamplifying step comprises ligase chain reaction or strand displacementamplification. In one aspect of this preferred embodiment, the bioagentis a bacterium, virus, cell or spore. Advantageously, the nucleic acidis ribosomal RNA. In another aspect, the nucleic acid encodes RNase P oran RNA-dependent RNA polymerase. Preferably, the amplification productis ionized prior to molecular mass determination. The method may furthercomprise the step of isolating nucleic acid from the bioagent prior tocontacting the nucleic acid with the at least one pair ofoligonucleotide primers. The method may further comprise the step ofperforming steps (a)-(d) using a different oligonucleotide primer pairand comparing the results to one or more molecular mass amplificationproducts obtained by performing steps (a)-(c) on a different pluralityof known organisms from those in step (d). Preferably, the one or moremolecular mass is contained in a database of molecular masses. Inanother aspect of this preferred embodiment, the amplification productis ionized by electrospray ionization, matrix-assisted laser desorptionor fast atom bombardment. Advantageously, the molecular mass isdetermined by mass spectrometry. Preferably, the mass spectrometry isFourier transform ion cyclotron resonance mass spectrometry (FT-ICR-MS),ion trap, quadrupole, magnetic sector, time of flight (TOF), Q-TOF ortriple quadrupole. The method may further comprise performing step (b)in the presence of an analog of adenine, thymidine, guanosine orcytidine having a different molecular weight than adenosine, thymidine,guanosine or cytidine. In one aspect, the oligonucleotide primercomprises a base analog or substitute base at positions 1 and 2 of eachtriplet within the primer, wherein the base analog or substitute basebinds with increased affinity to its complement compared to the nativebase. Preferably, the primer comprises a universal base at position 3 ofeach triplet within the primer. The base analog or substitute base maybe 2,6-diaminopurine, propyne T, propyne G, phenoxazines or G-clamp.Preferably, the universal base is inosine, guanidine, uridine,5-nitroindole, 3-nitropyrrole, dP or dK, or1-(2-deoxy-β-D-ribofuranosyl)-imidazole-4-carboxamide.

[0017] Another embodiment of the present invention is a method ofidentifying an unknown bioagent comprising (a) contacting nucleic acidfrom the bioagent with at least one pair of oligonucleotide primerswhich hybridize to sequences of the nucleic acid and flank a variablenucleic acid sequence; (b) amplifying the variable nucleic acid sequenceto produce an amplification product; (c) determining the basecomposition of the amplification product; and (d) comparing the basecomposition to one or more base compositions of amplification productsobtained by performing steps (a)-(c) on a plurality of known organisms,wherein a match identifies the unknown bioagent. In one aspect of thispreferred embodiment, the sequences to which the at least one pair ofoligonucleotide primers hybridize are highly conserved. Preferably, theamplifying step comprises polymerase chain reaction. Alternatively, theamplifying step comprises ligase chain reaction or strand displacementamplification. In one aspect of this preferred embodiment, the bioagentis a bacterium, virus, cell or spore. Advantageously, the nucleic acidis ribosomal RNA. In another aspect, the nucleic acid encodes RNase P oran RNA-dependent RNA polymerase. Preferably, the amplification productis ionized prior to molecular mass determination. The method may furthercomprise the step of isolating nucleic acid from the bioagent prior tocontacting the nucleic acid with the at least one pair ofoligonucleotide primers. The method may further comprise the step ofperforming steps (a)-(d) using a different oligonucleotide primer pairand comparing the results to one or more base composition signatures ofamplification products obtained by performing steps (a)-(c) on adifferent plurality of known organisms from those in step (d).Preferably, the one or more base compositions is contained in a databaseof base compositions. In another aspect of this preferred embodiment,the amplification product is ionized by electrospray ionization,matrix-assisted laser desorption or fast atom bombardment.Advantageously, the molecular mass is determined by mass spectrometry.Preferably, the mass spectrometry is Fourier transform ion cyclotronresonance mass spectrometry (FT-ICR-MS), ion trap, quadrupole, magneticsector, time of flight (TOF), Q-TOF or triple quadrupole. The method mayfurther comprise performing step (b) in the presence of an analog ofadenine, thymidine, guanosine or cytidine having a different molecularweight than adenosine, thymidine, guanosine or cytidine. In one aspect,the oligonucleotide primer comprises a base analog or substitute base atpositions 1 and 2 of each triplet within the primer, wherein the baseanalog or substitute base binds with increased affinity to itscomplement compared to the native base. Preferably, the primer comprisesa universal base at position 3 of each triplet within the primer. Thebase analog or substitute base may be 2,6-diaminopurine, propyne T,propyne G, phenoxazines or G-clamp. Preferably, the universal base isinosine, guanidine, uridine, 5-nitroindole, 3-nitropyrrole, dP or dK, or1-(2-deoxy-β-D-ribofuranosyl)-imidazole-4-carboxamide.

[0018] The present invention also provides a method for detecting asingle nucleotide polymorphism in an individual, comprising the steps of(a) isolating nucleic acid from the individual; (b) contacting thenucleic acid with oligonucleotide primers which hybridize to regions ofthe nucleic acid which flank a region comprising the potentialpolymorphism; (c) amplifying the region to produce an amplificationproduct; (d) determining the molecular mass of the amplificationproduct; and (e) comparing the molecular mass to the molecular mass ofthe region in an individual known to have the polymorphism, wherein ifthe molecular masses are the same then the individual has thepolymorphism.

[0019] In one aspect of this preferred embodiment, the primers hybridizeto highly conserved sequences. Preferably, the polymorphism isassociated with a disease. Alternatively, the polymorphism is a bloodgroup antigen. In one aspect of the preferred embodiment, the amplifyingstep is polymerase chain reaction. Alternatively, the amplification stepis ligase chain reaction or strand displacement amplification.Preferably, the amplification product is ionized prior to massdetermination. In one aspect, the amplification product is ionized byelectrospray ionization, matrix-assisted laser desorption or fast atombombardment. Advantageously, the molecular mass is determined by massspectrometry. Preferably, the mass spectrometry is Fourier transform ioncyclotron resonance mass spectrometry (FT-ICR-MS), ion trap, quadrupole,magnetic sector, time of flight (TOF), Q-TOF or triple quadrupole.

BRIEF DESCRIPTION OF THE DRAWINGS

[0020]FIGS. 1A-1I are consensus diagrams that show examples of conservedregions from 16S rRNA (FIG. 1A-1B), 23S rRNA (3′-half, FIG. 1C-1D;5′-half, FIG. 1E-F), 23S rRNA Domain I (FIG. 1G), 23S rRNA Domain IV(FIG. 1H) and 16S rRNA Domain III (FIG. 1I) which are suitable for usein the present invention. Lines with arrows are examples of regions towhich intelligent primer pairs for PCR are designed. The label for eachprimer pair represents the starting and ending base number of theamplified region on the consensus diagram. Bases in capital letters aregreater than 95% conserved; bases in lower case letters are 90-95%conserved, filled circles are 80-90% conserved; and open circles areless than 80% conserved. The label for each primer pair represents thestarting and ending base number of the amplified region on the consensusdiagram.

[0021]FIG. 2 shows a typical primer amplified region from the 16S rRNADomain III shown in FIG. 1C.

[0022]FIG. 3 is a schematic diagram showing conserved regions in RNaseP. Bases in capital letters are greater than 90% conserved; bases inlower case letters are 80-90% conserved; filled circles designate baseswhich are 70-80% conserved; and open circles designate bases that areless than 70% conserved.

[0023]FIG. 4 is a schematic diagram of base composition signaturedetermination using nucleotide analog “tags” to determine basecomposition signatures.

[0024]FIG. 5 shows the deconvoluted mass spectra of a Bacillus anthracisregion with and without the mass tag phosphorothioate A (A*). The twospectra differ in that the measured molecular weight of the masstag-containing sequence is greater than the unmodified sequence.

[0025]FIG. 6 shows base composition signature (BCS) spectra from PCRproducts from Staphylococcus aureus (S. aureus 16S_(—)1337F) andBacillus anthracus (B. anthr. 16S_(—)1337F), amplified using the sameprimers. The two strands differ by only two (AT→CG) substitutions andare clearly distinguished on the basis of their BCS.

[0026]FIG. 7 shows that a single difference between two sequences (A₁₄in B. anthracis vs. A₁₅ in B. cereus) can be easily detected usingESI-TOF mass spectrometry.

[0027]FIG. 8 is an ESI-TOF of Bacillus anthracis spore coat protein sspE56mer plus calibrant. The signals unambiguously identify B. anthracisversus other Bacillus species.

[0028]FIG. 9 is an ESI-TOF of a B. anthracis synthetic 16S_(—)1228duplex (reverse and forward strands). The technique easily distinguishesbetween the forward and reverse strands.

[0029]FIG. 10 is an ESI-FTICR-MS of a synthetic B. anthracis 16S_(—)133746 base pair duplex.

[0030]FIG. 11 is an ESI-TOF-MS of a 56mer oligonucleotide (3 scans) fromthe B. anthracis saspB gene with an internal mass standard. The internalmass standards are designated by asterisks.

[0031]FIG. 12 is an ESI-TOF-MS of an internal standard with 5 mM TBA-TFAbuffer showing that charge stripping with tributylammoniumtrifluoroacetate reduces the most abundant charge state from [M−8H⁺]⁸⁻to [M−3H⁺]³⁻.

DETAILED DESCRIPTION OF THE INVENTION

[0032] The present invention provides a combination of a non-PCR biomassdetection mode, preferably high-resolution MS, with PCR-based BCStechnology using “intelligent primers” which hybridize to conservedsequence regions of nucleic acids derived from a bioagent and whichbracket variable sequence regions that uniquely identify the bioagent.The high-resolution MS technique is used to determine the molecular massand base composition signature (BCS) of the amplified sequence region.This unique “base composition signature” (BCS) is then input to amaximum-likelihood detection algorithm for matching against a databaseof base composition signatures in the same amplified region. The presentmethod combines PCR-based amplification technology (which providesspecificity) and a molecular mass detection mode (which provides speedand does not require nucleic acid sequencing of the amplified targetsequence) for bioagent detection and identification.

[0033] The present method allows extremely rapid and accurate detectionand identification of bioagents compared to existing methods.Furthermore, this rapid detection and identification is possible evenwhen sample material is impure. Thus, the method is useful in a widevariety of fields, including, but not limited to, environmental testing(e.g., detection and discrimination of pathogenic vs. non-pathogenicbacteria in water or other samples), germ warfare (allowing immediateidentification of the bioagent and appropriate treatment),pharmacogenetic analysis and medical diagnosis (including cancerdiagnosis based on mutations and polymorphisms; drug resistance andsusceptibility testing; screening for and/or diagnosis of geneticdiseases and conditions; and diagnosis of infectious diseases andconditions). The method leverages ongoing biomedical research invirulence, pathogenicity, drug resistance and genome sequencing into amethod which provides greatly improved sensitivity, specificity andreliability compared to existing methods, with lower rates of falsepositives.

[0034] The present method can be used to detect and classify anybiological agent, including bacteria, viruses, fungi and toxins. As oneexample, where the agent is a biological threat, the informationobtained is used to determine practical information needed forcountermeasures, including toxin genes, pathogenicity islands andantibiotic resistance genes. In addition, the methods can be used toidentify natural or deliberate engineering events including chromosomefragment swapping, molecular breeding (gene shuffling) and emerginginfectious diseases.

[0035] Bacteria have a common set of absolutely required genes. About250 genes are present in all bacterial species (Proc. Natl. Acad. Sci.U.S.A. 93:10268, 1996; Science 270:397, 1995), including tiny genomeslike Mycoplasma, Ureaplasma and Rickettsia. These genes encode proteinsinvolved in translation, replication, recombination and repair,transcription, nucleotide metabolism, amino acid metabolism, lipidmetabolism, energy generation, uptake, secretion and the like. Examplesof these proteins are DNA polymerase III beta, elongation factor TU,heat shock protein groEL, RNA polymerase beta, phosphoglycerate kinase,NADH dehydrogenase, DNA ligase, DNA topoisomerase and elongation factorG. Operons can also be targeted using the present method. One example ofan operon is the bfp operon from enteropathogenic E. coli. Multiple corechromosomal genes can be used to classify bacteria at a genus or genusspecies level to determine if an organism has threat potential. Themethod can also be used to detect pathogenicity markers (plasmid orchromosomal) and antibiotic resistance genes to confirm the threatpotential of an organism and to direct countermeasures.

[0036] A theoretically ideal bioagent detector would identify, quantify,and report the complete nucleic acid sequence of every bioagent thatreached the sensor. The complete sequence of the nucleic acid componentof a pathogen would provide all relevant information about the threat,including its identity and the presence of drug-resistance orpathogenicity markers. This ideal has not yet been achieved. However,the present invention provides a straightforward strategy for obtaininginformation with the same practical value using base compositionsignatures (BCS). While the base composition of a gene fragment is notas information-rich as the sequence itself, there is no need to analyzethe complete sequence of the gene if the short analyte sequence fragmentis properly chosen. A database of reference sequences can be prepared inwhich each sequence is indexed to a unique base composition signature,so that the presence of the sequence can be inferred with accuracy fromthe presence of the signature. The advantage of base compositionsignatures is that they can be quantitatively measured in a massivelyparallel fashion using multiplex PCR (PCR in which two or more primerpairs amplify target sequences simultaneously) and mass spectrometry.These multiple primer amplified regions uniquely identify most threatand ubiquitous background bacteria and viruses. In addition,cluster-specific primer pairs distinguish important local clusters(e.g., anthracis group).

[0037] In the context of this invention, a “bioagent” is any organism,living or dead, or a nucleic acid derived from such an organism.Examples of bioagents include but are not limited to cells (includingbut not limited to human clinical samples, bacterial cells and otherpathogens) viruses, toxin genes and bioregulating compounds). Samplesmay be alive or dead or in a vegetative state (for example, vegetativebacteria or spores) and may be encapsulated or bioengineered.

[0038] As used herein, a

base composition signatures

(BCS) is the exact base composition from selected fragments of nucleicacid sequences that uniquely identifies the target gene and sourceorganism. BCS can be thought of as unique indexes of specific genes.

[0039] As used herein,

intelligent primers

are primers which bind to sequence regions which flank an interveningvariable region. In a preferred embodiment, these sequence regions whichflank the variable region are highly conserved among different speciesof bioagent. For example, the sequence regions may be highly conservedamong all Bacillus species. By the term

highly conserved

, it is meant that the sequence regions exhibit between about 80-100%,more preferably between about 90-100% and most preferably between about95-100% identity. Examples of intelligent primers which amplify regionsof the 16S and 23S rRNA are shown in FIGS. 1A-1I. A typical primeramplified region in 16S rRNA is shown in FIG. 2. The arrows representprimers which bind to highly conserved regions which flank a variableregion in 16S rRNA domain III. The amplified region is the stem-loopstructure under

1100-1188.

[0040] One main advantage of the detection methods of the presentinvention is that the primers need not be specific for a particularbacterial species, or even genus, such as Bacillus or Streptomyces.Instead, the primers recognize highly conserved regions across hundredsof bacterial species including, but not limited to, the speciesdescribed herein. Thus, the same primer pair can be used to identify anydesired bacterium because it will bind to the conserved regions whichflank a variable region specific to a single species, or common toseveral bacterial species, allowing nucleic acid amplification of theintervening sequence and determination of its molecular weight and basecomposition. For example, the 16S_(—)971-1062, 16S_(—)1228-1310 and16S_(—)1100-1188 regions are 98-99% conserved in about 900 species ofbacteria (16S=16S rRNA, numbers indicate nucleotide position). In oneembodiment of the present invention, primers used in the present methodbind to one or more of these regions or portions thereof.

[0041] The present invention provides a combination of a non-PCR biomassdetection mode, preferably high-resolution MS, with nucleic acidamplification-based BCS technology using “intelligent primers” whichhybridize to conserved regions and which bracket variable regions thatuniquely identify the bioagent(s). Although the use of PCR is preferred,other nucleic acid amplification techniques may also be used, includingligase chain reaction (LCR) and strand displacement amplification (SDA).The high-resolution MS technique allows separation of bioagent spectrallines from background spectral lines in highly cluttered environments.The resolved spectral lines are then translated to BCS which are inputto a maximum-likelihood detection algorithm matched against spectra forone or more known BCS. Preferably, the bioagent BCS spectrum is matchedagainst one or more databases of BCS from vast numbers of bioagents.Preferably, the matching is done using a maximum-likelihood detectionalgorithm.

[0042] In a preferred embodiment, base composition signatures arequantitatively measured in a massively parallel fashion using thepolymerase chain reaction (PCR), preferably multiplex PCR, and massspectrometric (MS) methods. Sufficient quantities of nucleic acids mustbe present for detection of bioagents by MS. A wide variety oftechniques for preparing large amounts of purified nucleic acids orfragments thereof are well known to those of skill in the art. PCRrequires one or more pairs of oligonucleotide primers which bind toregions which flank the target sequence(s) to be amplified. Theseprimers prime synthesis of a different strand of DNA, with synthesisoccurring in the direction of one primer towards the other primer. Theprimers, DNA to be amplified, a thermostable DNA polymerase (e.g. Taqpolymerase), the four deoxynucleotide triphosphates, and a buffer arecombined to initiate DNA synthesis. The solution is denatured byheating, then cooled to allow annealing of newly added primer, followedby another round of DNA synthesis. This process is typically repeatedfor about 30 cycles, resulting in amplification of the target sequence.

[0043] The “intelligent primers” define the target sequence region to beamplified and analyzed. In one embodiment, the target sequence is aribosomal RNA (rRNA) gene sequence. With the complete sequences of manyof the smallest microbial genomes now available, it is possible toidentify a set of genes that defines “minimal life” and identifycomposition signatures that uniquely identify each gene and organism.Genes that encode core life functions such as DNA replication,transcription, ribosome structure, translation, and transport aredistributed broadly in the bacterial genome and are preferred regionsfor BCS analysis. Ribosomal RNA (rRNA) genes comprise regions thatprovide useful base composition signatures. Like many genes involved incore life functions, rRNA genes contain sequences that areextraordinarily conserved across bacterial domains interspersed withregions of high variability that are more specific to each species. Thevariable regions can be utilized to build a database of base compositionsignatures. The strategy involves creating a structure-based alignmentof sequences of the small (16S) and the large (23S) subunits of the rRNAgenes. For example, there are currently over 13,000 sequences in theribosomal RNA database that has been created and maintained by RobinGutell, University of Texas at Austin, and is publicly available on theInstitute for Cellular and Molecular Biology web page(http://www.rna.icmb.utexas.edu/). There is also a publicly availablerRNA database created and maintained by the University of Antwerp,Belgium at http://www.rrna.uia.ac.be.

[0044] These databases have been analyzed to determine regions that areuseful as base composition signatures. The characteristics of suchregions are: a) between about 80 and 100%, preferably>about 95% identityamong species of the particular bioagent of interest, of upstream anddownstream nucleotide sequences which serve as sequence amplificationprimer sites; b) an intervening variable region which exhibits nogreater than about 5% identity among species; and c) a separation ofbetween about 30 and 1000 nucleotides, preferably no more than about50-250 nucleotides, and more preferably no more than about 60-100nucleotides, between the conserved regions.

[0045] Due to their overall conservation, the flanking rRNA primersequences serve as good “universal” primer binding sites to amplify theregion of interest for most, if not all, bacterial species. Theintervening region between the sets of primers varies in length and/orcomposition, and thus provides a unique base composition signature.

[0046] It is advantageous to design the “intelligent primers” to be asuniversal as possible to minimize the number of primers which need to besynthesized, and to allow detection of multiple species using a singlepair of primers. These primer pairs can be used to amplify variableregions in these species. Because any variation (due to codon wobble inthe 3^(rd) position) in these conserved regions among species is likelyto occur in the third position of a DNA triplet, oligonucleotide primerscan be designed such that the nucleotide corresponding to this positionis a base which can bind to more than one nucleotide, referred to hereinas a “universal base”. For example, under this “wobble” pairing, inosine(I) binds to U, C or A; guanine (G) binds to U or C, and uridine (U)binds to U or C. Other examples of universal bases include nitroindolessuch as 5-nitroindole or 3-nitropyrrole (Loakes et al., Nucleosides andNucleotides 14:1001-1003, 1995), the degenerate nucleotides dP or dK(Hill et al.), an acyclic nucleoside analog containing 5-nitroindazole(Van Aerschot et al., Nucleosides and Nucleotides 14:1053-1056, 1995) orthe purine analog 1-(2-deoxy-β-D-ribofuranosyl)-imidazole-4-carboxamide(Sala et al., Nucl. Acids Res. 24:3302-3306, 1996). In anotherembodiment of the invention, to compensate for the somewhat weakerbinding by the “wobble” base, the oligonucleotide primers are designedsuch that the first and second positions of each triplet are occupied bynucleotide analogs which bind with greater affinity than the unmodifiednucleotide. Examples of these analogs are 2,6-diaminopurine which bindsto thymine, propyne T which binds to adenine and propyne C andphenoxazines, including G-clamp, which binds to G. Propynes aredescribed in U.S. Pat. Nos. 5,645,985, 5,830,653 and 5,484,908, theentire contents of which are incorporated herein by reference.Phenoxazines are described in U.S. Pat. Nos. 5,502,177, 5,763,588, and6,005,096, the entire contents of which are incorporated herein byreference. G-clamps are described in U.S. Pat. Nos. 6,007,992 and6,028,183, the entire contents of which are incorporated herein byreference. Bacterial biological warfare agents capable of being detectedby the present methods include Bacillus anthracis (anthrax), Yersiniapestis (pneumonic plague), Franciscella tularensis (tularemia), Brucellasuis, Brucella abortus, Brucella melitensis (undulant fever),Burkholderia mallei (glanders), Burkholderia pseudomalleii(melioidosis), Salmonella typhi (typhoid fever), Rickettsia typhii(epidemic typhus), Rickettsia prowasekii (endemic typhus) and Coxiellaburnetii (Q fever), Rhodobacter capsulatus, Chlamydia pneumoniae,Escherichia coli, Shigella dysenteriae, Shigella flexneri, Bacilluscereus, Clostridium botulinum, Coxiella burnetti, Pseudomonasaeruginosa, Legionella pneumophila, and Vibrio cholerae.

[0047] Besides 16S and 23S rRNA, other target regions suitable for usein the present invention for detection of bacteria include 5S rRNA andRNase P (FIG. 3).

[0048] Biological warfare fungus biowarfare agents include coccidioidesimmitis (Coccidioidomycosis).

[0049] Biological warfare toxin genes capable of being detected by themethods of the present invention include botulism, T-2 mycotoxins,ricin, staph enterotoxin B, shigatoxin, abrin, aflatoxin, Clostridiumperfringens epsilon toxin, conotoxins, diacetoxyscirpenol, tetrodotoxinand saxitoxin. Biological warfare viral threat agents are mostly RNAviruses (positive-strand and negative-strand), with the exception ofsmallpox. Every RNA virus is a family of related viruses (quasispecies).These viruses mutate rapidly and the potential for engineered strains(natural or deliberate) is very high. RNA viruses cluster into familiesthat have conserved RNA structural domains on the viral genome (e.g.,virion components, accessory proteins) and conserved housekeeping genesthat encode core viral proteins including, for single strand positivestrand RNA viruses, RNA-dependent RNA polymerase, double stranded RNAhelicase, chymotrypsin-like and papain-like proteases andmethyltransferases.

[0050] Examples of (−)-strand RNA viruses include arenaviruses (e.g.,sabia virus, lassa fever, Machupo, Argentine hemorrhagic fever, flexalvirus), bunyaviruses (e.g., hantavirus, nairovirus, phlebovirus, hantaanvirus, Congo-crimean hemorrhagic fever, rift valley fever), andmononegavirales (e.g., filovirus, paramyxovirus, ebola virus, Marburg,equine morbillivirus).

[0051] Examples of (+)-strand RNA viruses include picornaviruses (e.g.,coxsackievirus, echovirus, human coxsackievirus A, human echovirus,human enterovirus, human poliovirus, hepatitis A virus, humanparechovirus, human rhinovirus), astroviruses (e.g., human astrovirus),calciviruses (e.g., chiba virus, chitta virus, human calcivirus, norwalkvirus), nidovirales (e.g., human coronavirus, human torovirus),flaviviruses (e.g., dengue virus 1-4, Japanese encephalitis virus,Kyanasur forest disease virus, Murray Valley encephalitis virus, Rociovirus, St. Louis encephalitis virus, West Nile virus, yellow fevervirus, hepatitis c virus) and togaviruses (e.g., Chikugunya virus,Eastern equine encephalitis virus, Mayaro virus, O'nyong-nyong virus,Ross River virus, Venezuelan equine encephalitis virus, Rubella virus,hepatitis E virus). The hepatitis C virus has a 5′-untranslated regionof 340 nucleotides, an open reading frame encoding 9 proteins having3010 amino acids and a 3′-untranslated region of 240 nucleotides. The5′-UTR and 3′-UTR are 99% conserved in hepatitis C viruses.

[0052] In one embodiment, the target gene is an RNA-dependent RNApolymerase or a helicase encoded by (+)-strand RNA viruses, or RNApolymerase from a (−)-strand RNA virus. (+)-strand RNA viruses aredouble stranded RNA and replicate by RNA-directed RNA synthesis usingRNA-dependent RNA polymerase and the positive strand as a template.Helicase unwinds the RNA duplex to allow replication of the singlestranded RNA. These viruses include viruses from the familypicornaviridae (e.g., poliovirus, coxsackievirus, echovirus),togaviridae (e.g., alphavirus, flavivirus, rubivirus), arenaviridae(e.g., lymphocytic choriomeningitis virus, lassa fever virus),cononaviridae (e.g., human respiratory virus) and Hepatitis A virus. Thegenes encoding these proteins comprise variable and highly conservedregions which flank the variable regions.

[0053] In a preferred embodiment, the detection scheme for the PCRproducts generated from the bioagent(s) incorporates three features.First, the technique simultaneously detects and differentiates multiple(generally about 6-10) PCR products. Second, the technique provides aBCS that uniquely identifies the bioagent from the possible primersites. Finally, the detection technique is rapid, allowing multiple PCRreactions to be run in parallel.

[0054] In one embodiment, the method can be used to detect the presenceof antibiotic resistance and/or toxin genes in a bacterial species. Forexample, Bacillus anthracis comprising a tetracycline resistance plasmidand plasmids encoding one or both anthracis toxins (px01 and/or px02)can be detected by using antibiotic resistance primer sets and toxingene primer sets. If the B. anthracis is positive for tetracyclineresistance, then a different antibiotic, for example quinalone, is used.

[0055] Mass spectrometry (MS)-based detection of PCR products providesall of these features with additional advantages. MS is intrinsically aparallel detection scheme without the need for radioactive orfluorescent labels, since every amplification product with a unique basecomposition is identified by its molecular mass. The current state ofthe art in mass spectrometry is such that less than femtomole quantitiesof material can be readily analyzed to afford information about themolecular contents of the sample. An accurate assessment of themolecular mass of the material can be quickly obtained, irrespective ofwhether the molecular weight of the sample is several hundred, or inexcess of one hundred thousand atomic mass units (amu) or Daltons.Intact molecular ions can be generated from amplification products usingone of a variety of ionization techniques to convert the sample to gasphase. These ionization methods include, but are not limited to,electrospray ionization (ES), matrix-assisted laser desorptionionization (MALDI) and fast atom bombardment (FAB). For example, MALDIof nucleic acids, along with examples of matrices for use in MALDI ofnucleic acids, are described in WO 98/54751 (Genetrace, Inc.).

[0056] Upon ionization, several peaks are observed from one sample dueto the formation of ions with different charges. Averaging the multiplereadings of molecular mass obtained from a single mass spectrum affordsan estimate of molecular mass of the bioagent. Electrospray ionizationmass spectrometry (ESI-MS) is particularly useful for very highmolecular weight polymers such as proteins and nucleic acids havingmolecular weights greater than 10 kDa, since it yields a distribution ofmultiply-charged molecules of the sample without causing a significantamount of fragmentation.

[0057] The mass detectors used in the methods of the present inventioninclude, but are not limited to, Fourier transform ion cyclotronresonance mass spectrometry (FT-ICR-MS), ion trap, quadrupole, magneticsector, time of flight (TOF), Q-TOF, and triple quadrupole.

[0058] In general, the mass spectrometric techniques which can be usedin the present invention include, but are not limited to, tandem massspectrometry, infrared multiphoton dissociation and pyrolytic gaschromatography mass spectrometry (PGC-MS). In one embodiment of theinvention, the bioagent detection system operates continually inbioagent detection mode using pyrolytic GC-MS without PCR for rapiddetection of increases in biomass (for example, increases in fecalcontamination of drinking water or of germ warfare agents). To achieveminimal latency, a continuous sample stream flows directly into thePGC-MS combustion chamber. When an increase in biomass is detected, aPCR process is automatically initiated. Bioagent presence produceselevated levels of large molecular fragments from 100-7,000 Da which areobserved in the PGC-MS spectrum. The observed mass spectrum is comparedto a threshold level and when levels of biomass are determined to exceeda predetermined threshold, the bioagent classification process describedhereinabove(combining PCR and MS, preferably FT-ICR MS) is initiated.Optionally, alarms or other processes (halting ventilation flow,physical isolation) are also initiated by this detected biomass level.

[0059] The accurate measurement of molecular mass for large DNAs islimited by the adduction of cations from the PCR reaction to eachstrand, resolution of the isotopic peaks from natural abundance ¹³C and¹⁵N isotopes, and assignment of the charge state for any ion. Thecations are removed by in-line dialysis using a flow-through chip thatbrings the solution containing the PCR products into contact with asolution containing ammonium acetate in the presence of an electricfield gradient orthogonal to the flow. The latter two problems areaddressed by operating with a resolving power of >100,000 and byincorporating isotopically depleted nucleotide triphosphates into theDNA. The resolving power of the instrument is also a consideration. At aresolving power of 10,000, the modeled signal from the [M−14H+]¹⁴⁻charge state of an 84mer PCR product is poorly characterized andassignment of the charge state or exact mass is impossible. At aresolving power of 33,000, the peaks from the individual isotopiccomponents are visible. At a resolving power of 100,000, the isotopicpeaks are resolved to the baseline and assignment of the charge statefor the ion is straightforward. The [¹³C, ¹⁵N]-depleted triphosphatesare obtained, for example, by growing microorganisms on depleted mediaand harvesting the nucleotides (Batey et al., Nucl. Acids Res.20:4515-4523, 1992).

[0060] While mass measurements of intact nucleic acid regions arebelieved to be adequate to determine most bioagents, tandem massspectrometry (MS^(n)) techniques may provide more definitive informationpertaining to molecular identity or sequence. Tandem MS involves thecoupled use of two or more stages of mass analysis where both theseparation and detection steps are based on mass spectrometry. The firststage is used to select an ion or component of a sample from whichfurther structural information is to be obtained. The selected ion isthen fragmented using, e.g., blackbody irradiation, infrared multiphotondissociation, or collisional activation. For example, ions generated byelectrospray ionization (ESI) can be fragmented using IR multiphotondissociation. This activation leads to dissociation of glycosidic bondsand the phosphate backbone, producing two series of fragment ions,called the w-series (having an intact 3′ terminus and a 5′ phosphatefollowing internal cleavage) and the a-Base series(having an intact 5′terminus and a 3′ furan).

[0061] The second stage of mass analysis is then used to detect andmeasure the mass of these resulting fragments of product ions. Such ionselection followed by fragmentation routines can be performed multipletimes so as to essentially completely dissect the molecular sequence ofa sample.

[0062] If there are two or more targets of similar base composition ormass, or if a single amplification reaction results in a product whichhas the same mass as two or more bioagent reference standards, they canbe distinguished by using mass-modifying “tags.” In this embodiment ofthe invention, a nucleotide analog or “tag” is incorporated duringamplification (e.g., a 5-(trifluoromethyl)deoxythymidine triphosphate)which has a different molecular weight than the unmodified base so as toimprove distinction of masses. Such tags are described in, for example,PCT WO97/33000. This further limits the number of possible basecompositions consistent with any mass. For example,5-(trifluoromethyl)deoxythymidine triphosphate can be used in place ofdTTP in a separate nucleic acid amplification reaction. Measurement ofthe mass shift between a conventional amplification product and thetagged product is used to quantitate the number of thymidine nucleotidesin each of the single strands. Because the strands are complementary,the number of adenosine nucleotides in each strand is also determined.

[0063] In another amplification reaction, the number of G and C residuesin each strand is determined using, for example, the cytidine analog5-methylcytosine (5-meC) or propyne C. The combination of the A/Treaction and G/C reaction, followed by molecular weight determination,provides a unique base composition. This method is summarized in FIG. 4and Table 1. TABLE 1 Total Total Total Base Base base base Double Single)mass info info comp. comp. strand strand this this other Top BottomMass tag sequence sequence strand strand strand strand strand T*)massT*ACGT*ACGT* T*ACGT*ACGT* 3x 3T 3A 3T 3A (T* − T) = x AT*GCAT*GCA 2A 2T2C 2G 2G 2C AT*GCAT*GCA 2x 2T 2A C*)mass TAC*GTAC*GT TAC*GTAC*GT 2x 2C2G (C* − C) = y ATGC*ATGC*A ATGC*ATGC*A 2x 2C 2G

[0064] The mass tag phosphorothioate A (A*) was used to distinguish aBacillus anthracis cluster. The B. anthracis (A₁₄G₉C₁₄T₉) had an averageMW of 14072.26, and the B. anthracis (A₁A*₁₃G₉C₁₄T₉) had an averagemolecular weight of 14281.11 and the phosphorothioate A had an averagemolecular weight of +16.06 as determined by ESI-TOF MS. The deconvolutedspectra are shown in FIG. 5.

[0065] In another example, assume the measured molecular masses of eachstrand are 30,000.115 Da and 31,000.115 Da respectively, and themeasured number of dT and dA residues are (30,28) and (28,30). If themolecular mass is accurate to 100 ppm, there are 7 possible combinationsof dG+dC possible for each strand. However, if the measured molecularmass is accurate to 10 ppm, there are only 2 combinations of dG+dC, andat 1 ppm accuracy there is only one possible base composition for eachstrand.

[0066] Signals from the mass spectrometer may be input to amaximum-likelihood detection and classification algorithm such as iswidely used in radar signal processing. The detection processing usesmatched filtering of BCS observed in mass-basecount space and allows fordetection and subtraction of signatures from known, harmless organisms,and for detection of unknown bioagent threats. Comparison of newlyobserved bioagents to known bioagents is also possible, for estimationof threat level, by comparing their BCS to those of known organisms andto known forms of pathogenicity enhancement, such as insertion ofantibiotic resistance genes or toxin genes.

[0067] Processing may end with a Bayesian classifier using loglikelihood ratios developed from the observed signals and averagebackground levels. The program emphasizes performance predictionsculminating in probability-of-detection versusprobability-of-false-alarm plots for conditions involving complexbackgrounds of naturally occurring organisms and environmentalcontaminants. Matched filters consist of a priori expectations of signalvalues given the set of primers used for each of the bioagents. Agenomic sequence database (e.g. GenBank) is used to define the massbasecount matched filters. The database contains known threat agents andbenign background organisms. The latter is used to estimate and subtractthe signature produced by the background organisms. A maximum likelihooddetection of known background organisms is implemented using matchedfilters and a running-sum estimate of the noise covariance. Backgroundsignal strengths are estimated and used along with the matched filtersto form signatures which are then subtracted. the maximum likelihoodprocess is applied to this “cleaned up” data in a similar manneremploying matched filters for the organisms and a running-sum estimateof the noise-covariance for the cleaned up data.

[0068] In one embodiment, a strategy to “triangulate” each organism bymeasuring signals from multiple core genes is used to reduce falsenegative and false positive signals, and enable reconstruction of theorigin or hybrid or otherwise engineered bioagents. After identificationof multiple core genes, alignments are created from nucleic acidsequence databases. The alignments are then analyzed for regions ofconservation and variation, and potential primer binding sites flankingvariable regions are identified. Next, amplification target regions forsignature analysis are selected which distinguishes organisms based onspecific genomic differences (i.e., base composition). For example,detection of signatures for the three part toxin genes typical of B.anthracis (Bowen, J. E. and C. P. Quinn, J. Appl. Microbiol. 1999, 87,270-278) in the absence of the expected signatures from the B. anthracisgenome would suggest a genetic engineering event.

[0069] The present method can also be used to detect single nucleotidepolymorphisms (SNPs), or multiple nucleotide polymorphisms, rapidly andaccurately. A SNP is defined as a single base pair site in the genomethat is different from one individual to another. The difference can beexpressed either as a deletion, an insertion or a substitution, and isfrequently linked to a disease state. Because they occur every 100-1000base pairs, SNPs are the most frequently bound type of genetic marker inthe human genome.

[0070] For example, sickle cell anemia results from an A-T transition,which encodes a valine rather than a glutamic acid residue.Oligonucleotide primers may be designed such that they bind to sequenceswhich flank a SNP site, followed by nucleotide amplification and massdetermination of the amplified product. Because the molecular masses ofthe resulting product from an individual who does not have sickle cellanemia is different from that of the product from an individual who hasthe disease, the method can be used to distinguish the two individuals.Thus, the method can be used to detect any known SNP in an individualand thus diagnose or determine increased susceptibility to a disease orcondition.

[0071] In one embodiment, blood is drawn from an individual andperipheral blood mononuclear cells (PBMC) are isolated andsimultaneously tested, preferably in a high-throughput screening method,for one or more SNPs using appropriate primers based on the knownsequences which flank the SNP region. The National Center forBiotechnology Information maintains a publicly available database ofSNPs (http://www.ncbi.nlm.nih.gov/SNP/).

[0072] The method of the present invention can also be used for bloodtyping. The gene encoding A, B or O blood type can differ by four singlenucleotide polymorphisms. If the gene contains the sequenceCGTGGTGACCCTT, antigen A results. If the gene contains the sequenceCGTCGTCACCGCTA antigen B results. If the gene contains the sequenceCGTGGT-ACCCCTT, blood group O results (“-” indicates a deletion). Thesesequences can be distinguished by designing a single primer pair whichflanks these regions, followed by amplification and mass determination.

[0073] While the present invention has been described with specificityin accordance with certain of its preferred embodiments, the followingexamples serve only to illustrate the invention and are not intended tolimit the same.

EXAMPLE 1 Nucleic Acid Isolation and PCR

[0074] In one embodiment, nucleic acid is isolated from the organismsand amplified by PCR using standard methods prior to BCS determinationby mass spectrometry. Nucleic acid is isolated, for example, bydetergent lysis of bacterial cells, centrifugation and ethanolprecipitation. Nucleic acid isolation methods are described in, forexample, Current Protocols in Molecular Biology (Ausubel et al.) andMolecular Cloning; A Laboratory Manual (Sambrook et al.). The nucleicacid is then amplified using standard methodology, such as PCR, withprimers which bind to conserved regions of the nucleic acid whichcontain an intervening variable sequence as described below.

EXAMPLE 2 Mass Spectrometry

[0075] FTICR Instrumentation: The FTICR instrument is based on a 7 teslaactively shielded superconducting magnet and modified Bruker DaltonicsApex II 70e ion optics and vacuum chamber. The spectrometer isinterfaced to a LEAP PAL autosampler and a custom fluidics controlsystem for high throughput screening applications. Samples are analyzeddirectly from 96-well or 384-well microtiter plates at a rate of about 1sample/minute. The Bruker data-acquisition platform is supplemented witha lab-built ancillary NT datastation which controls the autosampler andcontains an arbitrary waveform generator capable of generating complexrf-excite waveforms (frequency sweeps, filtered noise, stored waveforminverse Fourier transform (SWIFT), etc.) for sophisticated tandem MSexperiments. For oligonucleotides in the 20-30-mer regime typicalperformance characteristics include mass resolving power in excess of100,000 (FWHM), low ppm mass measurement errors, and an operable m/zrange between 50 and 5000 m/z.

[0076] Modified ESI Source: In sample-limited analyses, analytesolutions are delivered at 150 nL/minute to a 30 mm i.d. fused-silicaESI emitter mounted on a 3-D micromanipulator. The ESI ion opticsconsist of a heated metal capillary, an rf-only hexapole, a skimmercone, and an auxiliary gate electrode. The 6.2 cm rf-only hexapole iscomprised of 1 mm diameter rods and is operated at a voltage of 380 Vppat a frequency of 5 MHz. A lab-built electromechanical shutter can beemployed to prevent the electrospray plume from entering the inletcapillary unless triggered to the “open” position via a TTL pulse fromthe data station. When in the “closed” position, a stable electrosprayplume is maintained between the ESI emitter and the face of the shutter.The back face of the shutter arm contains an elastomeric seal which canbe positioned to form a vacuum seal with the inlet capillary. When theseal is removed, a 1 mm gap between the shutter blade and the capillaryinlet allows constant pressure in the external ion reservoir regardlessof whether the shutter is in the open or closed position. When theshutter is triggered, a “time slice” of ions is allowed to enter theinlet capillary and is subsequently accumulated in the external ionreservoir. The rapid response time of the ion shutter (<25 ms) providesreproducible, user defined intervals during which ions can be injectedinto and accumulated in the external ion reservoir.

[0077] Apparatus for Infrared Multiphoton Dissociation A 25 watt CW CO₂laser operating at 10.6 μm has been interfaced to the spectrometer toenable infrared multiphoton dissociation (IRMPD) for oligonucleotidesequencing and other tandem MS applications. An aluminum optical benchis positioned approximately 1.5 m from the actively shieldedsuperconducting magnet such that the laser beam is aligned with thecentral axis of the magnet. Using standard IR-compatible mirrors andkinematic mirror mounts, the unfocused 3 mm laser beam is aligned totraverse directly through the 3.5 mm holes in the trapping electrodes ofthe FTICR trapped ion cell and longitudinally traverse the hexapoleregion of the external ion guide finally impinging on the skimmer cone.This scheme allows IRMPD to be conducted in an m/z selective manner inthe trapped ion cell (e.g. following a SWIFT isolation of the species ofinterest), or in a broadband mode in the high pressure region of theexternal ion reservoir where collisions with neutral molecules stabilizeIRMPD-generated metastable fragment ions resulting in increased fragmention yield and sequence coverage.

EXAMPLE 3 Identification of Bioagents

[0078] Table 1 shows a small cross section of a database of calculatedmolecular masses for over 9 primer sets and approximately 30 organisms.The primer sets were derived from rRNA alignment. Examples of regionsfrom rRNA consensus alignments are shown in FIGS. 1A-1C. Lines witharrows are examples of regions to which intelligent primer pairs for PCRare designed. The primer pairs are >95% conserved in the bacterialsequence database (currently over 10,000 organisms). The interveningregions are variable in length and/or composition, thus providing thebase composition “signature” (BCS) for each organism. Primer pairs werechosen so the total length of the amplified region is less than about80-90 nucleotides. The label for each primer pair represents thestarting and ending base number of the amplified region on the consensusdiagram.

[0079] Included in the short bacterial database cross-section in Table 1are many well known pathogens/biowarfare agents (shown in bold/redtypeface) such as Bacillus anthracis or Yersinia pestis as well as someof the bacterial organisms found commonly in the natural environmentsuch as Streptomyces. Even closely related organisms can bedistinguished from each other by the appropriate choice of primers. Forinstance, two low G+C organisms, Bacillus anthracis and Staph aureus,can be distinguished from each other by using the primer pair defined by16S_(—)1337 or 23S_(—)855 ( )M of 4 Da). TABLE 2 Cross Section Of ADatabase Of Calculated Molecular Masses¹ Primer Regions Bug Name 16S_97116S_1100 16S_1337 16S_1294 16S_1228 23S_1021 23S_855 23S_193 23S_115Acinetobacter calcoaceticus 55619.1 55004 28446.7 35854.9 51295.4 3029942654 39557.5 54999

55005 54388 28448 35238 51296 30295 42651 39560 56850 Bacillus cereus55622.1 54387.9 28447.6 35854.9 51296.4 30295 42651 39560.5 56850.3Bordetella bronchiseptica 56857.3 51300.4 28446.7 35857.9 51307.4 3029942653 39559.5 51920.5 Borrelia burgdorferi 56231.2 55621.1 28440.735852.9 51295.4 30297 42029.9 38941.4 52524.6

58098 55011 28448 35854 50683 Campylobacter jejuni 58088.5 54386.929061.8 35856.9 50674.3 30294 42032.9 39558.5 45732.5

55000 55007 29063 35855 50676 30295 42036 38941 56230

55006 53767 28445 35855 51291 30300 42656 39562 54999 Clostridiumdifficile 56855.3 54386.9 28444.7 35853.9 51296.4 30294 41417.8 39556.555612.2 Enterococcus faecalis 55620.1 54387.9 28447.6 35858.9 51296.430297 42652 39559.5 56849.3

55622 55009 28445 35857 51301 30301 42656 39562 54999

53769 54385 28445 35856 51298 Haemophilus influenzae 55620.1 5500628444.7 35855.9 51298.4 30298 42656 39560.5 55613.1 Kiebsiellapneumoniae 55622.1 55008 28442.7 35856.9 51297.4 30300 42655 39562.555000

55618 55626 28446 35857 51303 Mycobacterium avium 54390.9 55631.129064.8 35858.9 51915.5 30298 42656 38942.4 56241.2 Mycobacterium leprae54389.9 55629.1 29064.8 35860.9 51917.5 30298 42656 39559.5 56240.2Mycobacterium tuberculosis 54390.9 55629.1 29064.8 35860.9 51301.4 3029942656 39560.5 56243.2 Mycoplasma genitalium 53143.7 45115.4 29061.835854.9 50671.3 30294 43264.1 39558.5 56842.4 Mycoplasma pneumoniae53143.7 45118.4 29061.8 35854.9 50673.3 30294 43264.1 39559.5 56843.4Neisseria gonorrhoeae 55627.1 54389.9 28445.7 35855.9 51302.4 3030042649 39561.5 55000

55623 55010 28443 35858 51301 30298 43272 39558 55619

58093 55621 28448 35853 50677 30293 42650 39559 53139

58094 55623 28448 35853 50679 30293 42648 39559 53755

55622 55005 28445 35857 51301 30301 42658

55623 55009 28444 35857 51301 Staphylococcus aureus 56854.3 54386.928443.7 35852.9 51294.4 30298 42655 39559.5 57466.4 Streptomyces 54389.959341.6 29063.8 35858.9 51300.4 39563.5 56864.3 Treponema pallidum56245.2 55631.1 28445.7 35851.9 51297.4 30299 42034.9 38939.4 57473.4

55625 55626 28443 35857 52536 29063 30303 35241 50675 Vibrioparahaemolyticus 54384.9 55626.1 28444.7 34620.7 50064.2

55620 55626 28443 35857 51299

[0080]FIG. 6 shows the use of ESI-FT-ICR MS for measurement of exactmass. The spectra from 46mer PCR products originating at position 1337of the 16S rRNA from S. aureus (upper) and B. anthracis (lower) areshown. These data are from the region of the spectrum containing signalsfrom the [M−8H+]⁸⁻ charge states of the respective 5′-3′ strands. Thetwo strands differ by two (AT-CG) substitutions, and have measuredmasses of 14206.396 and 14208.373±0.010 Da, respectively. The possiblebase compositions derived from the masses of the forward and reversestrands for the B. anthracis products are listed in Table 3. TABLE 3Possible base composition for B. anthracis products Calc. Mass ErrorBase Comp. 14208.2935 0.079520 A1 G17 C10 T18 14208.3160 0.056980 A1 G20C15 T10 14208.3386 0.034440 A1 G23 C20 T2 14208.3074 0.065560 A6 G11 C3T26 14208.3300 0.043020 A6 G14 C8 T18 14208.3525 0.020480 A6 G17 C13 T1014208.3751 0.002060 A6 G20 C18 T2 14208.3439 0.029060 A11 G8 C1 T2614208.3665 0.006520 A11 G11 C6 T18 14208.3890 0.016020 A11 G14 C11 T1014208.4116 0.038560 A11 G17 C16 T2 14208.4030 0.029980 A16 G8 C4 T1814208.4255 0.052520 A16 G11 C9 T10 14208.4481 0.075060 A16 G14 C14 T214208.4395 0.066480 A21 G5 C2 T18 14208.4620 0.089020 A21 G8 C7 T1014079.2624 0.080600 A0 G14 C13 T19 14079.2849 0.058060 A0 G17 C18 T1114079.3075 0.035520 A0 G20 C23 T3 14079.2538 0.089180 A5 G5 C1 T3514079.2764 0.066640 A5 G8 C6 T27 14079.2989 0.044100 A5 G11 C11 T1914079.3214 0.021560 A5 G14 C16 T11 14079.3440 0.000980 A5 G17 C21 T314079.3129 0.030140 A10 G5 C4 T27 14079.3354 0.007600 A10 G8 C9 T1914079.3579 0.014940 A10 G11 C14 T11 14079.3805 0.037480 A10 G14 C19 T314079.3494 0.006360 A15 G2 C2 T27 14079.3719 0.028900 A15 G5 C7 T1914079.3944 0.051440 A15 G8 C12 T11 14079.4170 0.073980 A15 G11 C17 T314079.4084 0.065400 A20 G2 C5 T19 14079.4309 0.087940 A20 G5 C10 T13

[0081] Among the 16 compositions for the forward strand and the 18compositions for the reverse strand that were calculated, only one pair(shown in bold) are complementary, corresponding to the actual basecompositions of the B. anthracis PCR products.

EXAMPLE 4 BCS of Region from Bacillus anthracis and Bacillus cereus

[0082] A conserved Bacillus region from B. anthracis (A₁₄G₉C₁₄T₉) and B.cereus (A₁₅G₉C₁₃T₉) having a C to A base change was synthesized andsubjected to ESI-TOF MS. The results are shown in FIG. 7 in which thetwo regions are clearly distinguished using the method of the presentinvention (MW=14072.26 vs. 14096.29).

EXAMPLE 5 Identification of Additional Bioagents

[0083] In other examples of the present invention, the pathogen Vibriocholera can be distinguished from Vibrio parahemolyticus with ΔM>600 Dausing one of three 16S primer sets shown in Table 2 (16S_(—)971,16S_(—)1228 or 16S_(—)1294) as shown in Table 4. The two mycoplasmaspecies in the list (M. genitalium and M. pneumoniae) can also bedistinguished from each other, as can the three mycobacteriae. While thedirect mass measurements of amplified products can identify anddistinguish a large number of organisms, measurement of the basecomposition signature provides dramatically enhanced resolving power forclosely related organisms. In cases such as Bacillus anthracis andBacillus cereus that are virtually indistinguishable from each otherbased solely on mass differences, compositional analysis orfragmentation patterns are used to resolve the differences. The singlebase difference between the two organisms yields different fragmentationpatterns, and despite the presence of the ambiguous/unidentified base Nat position 20 in B. anthracis, the two organisms can be identified.

[0084] Tables 4a-b show examples of primer pairs from Table 1 whichdistinguish pathogens from background. TABLE 4a Organism name 23S_85516S_1337 23S_1021 Bacillus anthracis 42650.98 28447.65 30294.98Staphylococcus aureus 42654.97 28443.67 30297.96

[0085] TABLE 4b Organism name 16S_971 16S_1294 16S_1228 Vibrio cholerae55625.09 35856.87 52535.59 Vibrio parahaemolyticus 54384.91 34620.6750064.19

[0086] Table 4 shows the expected molecular weight and base compositionof region 16S_(—)1100-1188 in Mycobacterium avium and Streptomyces sp.TABLE 5 Molecular Region Organism name Length weight Base comp.16S_1100- Mycobacterium 82 25624.1728 A₁₆G₃₂C₁₈T₁₆ 1188 avium 16S_1100-Streptomyces 96 29904.871 A₁₇G₃₈C₂₇T₁₄ 1188 sp.

[0087] Table 5 shows base composition (single strand) results for16S_(—)1100-1188 primer amplification reactions different species ofbacteria. Species which are repeated in the table (e.g., Clostridiumbotulinum) are different strains which have different base compositionsin the 16S_(—)1100-1188 region. TABLE 6 Organism name Base comp.Organism name Base comp. Mycobacterium avium A₁₆G₃₂C₁₈T₁₆ Vibriocholerae A₂₃G₃₀C₂₁T₁₆ Streptomyces sp. A₁₇G₃₈C₂₇T₁₄

A ₂₃ G ₃₁ C ₂₁ T ₁₅ Ureaplasma urealyticum A₁₈G₃₀C₁₇T₁₇

A ₂₃ G ₃₁ C ₂₁ T ₁₅ Streptomyces sp. A₁₉G₃₆C₂₄T₁₈ Mycoplasma genitaliumA₂₄G₁₉C₁₂T₁₈ Mycobacterium leprae A₂₀G₃₂C₂₂T₁₆ Clostridium botulinumA₂₄G₂₅C₁₈T₂₀

A ₂₀ G ₃₃ C ₂₁ T ₁₆ Bordetella bronchiseptica A₂₄G₂₆C₁₉T₁₄

A ₂₀ G ₃₃ C ₂₁ T ₁₆ Francisella tularensis A₂₄G₂₆C₁₉T₁₉ Fusobacteriumnecroforum A₂₁G₂₆C₂₂T₁₆

A ₂₄ G ₂₆ C ₂₀ T ₁₈ Listeria monocytogenes A₂₁G₂₇C₁₉T₁₉

A ₂₄ G ₂₆ C ₂₀ T ₁₈ Clostridium botulinum A₂₁G₂₇C₁₉T₂₁

A ₂₄ G ₂₆ C ₂₀ T ₁₈ Neisseria gonorrhoeae A₂₁G₂₈C₂₁T₁₈ Helicobacterpylori A₂₄G₂₆C₂₀T₁₉ Bartonella quintana A₂₁G₃₀C₂₂T₁₆ Helicobacter pyloriA₂₄G₂₆C₂₁T₁₈ Enterococcus faecalis A₂₂G₂₇C₂₀T₁₉ Moraxella catarrhalisA₂₄G₂₆C₂₃T₁₆ Bacillus megaterium A₂₂G₂₈C₂₀T₁₈ Haemophilus influenzae RdA₂₄G₂₈C₂₀T₁₇ Bacillus subtilis A₂₂G₂₈C₂₁T₁₇

A ₂₄ G ₂₈ C ₂₁ T ₁₆ Pseudomonas aeruginosa A₂₂G₂₉C₂₃T₁₅

A ₂₄ G ₂₈ C ₂₁ T ₁₆ Legionella pneumophila A₂₂G₃₂C₂₀T₁₆

A ₂₄ G ₂₈ C ₂₁ T ₁₆ Mycoplasma pneumoniae A₂₃G₂₀C₁₄T₁₆ Pseudomonasputida A₂₄G₂₉C₂₁T₁₆ Clostridium botulinum A₂₃G₂₆C₂₀T₁₉

A ₂₄ G ₃₀ C ₂₁ T ₁₅ Enterococcus faecium A₂₃G₂₆C₂₁T₁₈

A ₂₄ G ₃₀ C ₂₁ T ₁₅ Acinetobacter calcoaceti A₂₃G₂₆C₂₁T₁₉

A ₂₄ G ₃₀ C ₂₁ T ₁₅

A ₂₃ G ₂₆ C ₂₄ T ₁₅ Clostridium botulinum A₂₅G₂₄C₁₈T₂₁

A ₂₃ G ₂₆ C ₂₄ T ₁₅ Clostridium tetani A₂₅G₂₅C₁₈T₂₀ Clostridiumperfringens A₂₃G₂₇C₁₉T₁₉ Francisella tularensis A₂₅G₂₅C₁₉T₁₉

A ₂₃ G ₂₇ C ₂₀ T ₁₈ Acinetobacter calcoacetic A₂₅G₂₆C₂₀T₁₉

A ₂₃ G ₂₇ C ₂₀ T ₁₈ Bacteriodes fragilis A₂₅G₂₇C₁₆T₂₂

A ₂₃ G ₂₇ C ₂₀ T ₁₈ Chlamydophila psittaci A₂₅G₂₇C₂₁T₁₆ Aeromonashydrophila A₂₃G₂₉C₂₁T₁₆ Borrelia burgdorferi A₂₅G₂₉C₁₇T₁₉ Escherichiacoli A₂₃G₂₉C₂₁T₁₆ Streptobacillus monilifor A₂₆G₂₆C₂₀T₁₆ Pseudomonasputida A₂₃G₂₉C₂₁T₁₇ Rickettsia prowazekii A₂₆G₂₈C₁₈T₁₈

A ₂₃ G ₂₉ C ₂₂ T ₁₅ Rickettsia rickettsii A₂₆G₂₈C₂₀T₁₆

A ₂₃ G ₂₉ C ₂₂ T ₁₅ Mycoplasma mycoides A₂₈G₂₃C₁₆T₂₀

[0088] The same organism having different base compositions aredifferent strains. Groups of organisms which are highlighted or initalics have the same base compositions in the amplified region. Some ofthese organisms can be distinguished using multiple primers. Forexample, Bacillus anthracis can be distinguished from Bacillus cereusand Bacillus thuringiensis using the primer 16S_(—)971-1062 (Table 6).Other primer pairs which produce unique base composition signatures areshown in Table 6 (bold). Clusters containing very similar threat andubiquitous non-threat organisms (e.g. anthracis cluster) aredistinguished at high resolution with focused sets of primer pairs. Theknown biowarfare agents in Table 6 are Bacillus anthracis, Yersiniapestis, Francisella tularensis and Rickettsia prowazekii. TABLE 716S_971- 16S_1228- 16S_1100- Organism 1062 1310 1188 AeromonasA₂₁G₂₉C₂₂T₂₀ A₂₂G₂₇C₂₁T₁₃ A₂₃G₃₁C₂₁T₁₅ hydrophila Aeromonas A₂₁G₂₉C₂₂T₂₀A₂₂G₂₇C₂₁T₁₃ A₂₃G₃₁C₂₁T₁₅ salmonicida Bacillus anthracis A ₂₁G₂₇C₂₂T₂₂A₂₄G₂₂C₁₉T₁₈ A₂₃G₂₇C₂₀T₁₈ Bacillus cereus A₂₂G₂₇C₂₁T₂₂ A₂₄G₂₂C₁₉T₁₈A₂₃G₂₇C₂₀T₁₈ Bacillus A₂₂G₂₇C₂₁T₂₂ A₂₄G₂₂C₁₉T₁₈ A₂₃G₂₇C₂₀T₁₈thuringiensis Chlamydia A ₂₂G₂₆C₂₀T₂₃ A ₂₄G₂₃C₁₉T₁₆ A₂₄G₂₈C₂₁T₁₆trachomatis Chlamydia A₂₆G₂₃C₂₀T₂₂ A₂₆G₂₂C₁₆T₁₈ A₂₄G₂₈C₂₁T₁₆ pneumoniaeAR39 Leptospira A₂₂G₂₆C₂₀T₂₁ A₂₂G₂₅C₂₁T₁₅ A₂₃G₂₆C₂₄T₁₅ borgpeterseniiLeptospira A₂₂G₂₆C₂₀T₂₁ A₂₂G₂₅C₂₁T₁₅ A₂₃G₂₆C₂₄T₁₅ interrogans MycoplasmaA₂₈G₂₃C₁₅T₂₂ A ₃₀G₁₈C₁₅T₁₉ A ₂₄G₁₉C₁₂T₁₈ genitalium MycoplasmaA₂₈G₂₃C₁₅T₂₂ A ₂₇G₁₉C₁₆T₂₀ A ₂₃G₂₀C₁₄T₁₆ pneumoniae Escherichia coli A₂₂G₂₈C₂₀T₂₂ A₂₄G₂₅C₂₁T₁₃ A₂₃G₂₉C₂₂T₁₅ Shigella A ₂₂G₂₈C₂₁T₂₁A₂₄G₂₅C₂₁T₁₃ A₂₃G₂₉C₂₂T₁₅ dysenteriae Proteus vulgaris A ₂₃G₂₆C₂₂T₂₁ A₂₆G₂₄C₁₉T₁₄ A₂₄G₃₀C₂₁T₁₅ Yersinia pestis A₂₄G₂₅C₂₁T₂₂ A₂₅G₂₄C₂₀T₁₄A₂₄G₃₀C₂₁T₁₅ Yersinia A₂₄G₂₅C₂₁T₂₂ A₂₅G₂₄C₂₀T₁₄ A₂₄G₃₀C₂₁T₁₅pseudotuberculosis Francisella A ₂₀G₂₅C₂₁T₂₃ A ₂₃G₂₆C₁₇T₁₇ A ₂₄G₂₆C₁₉T₁₉tularensis Rickettsia A ₂₁G₂₆C₂₄T₂₅ A ₂₄G₂₃C₁₆T₁₉ A ₂₆G₂₈C₁₈T₁₈prowazekii Rickettsia A ₂₁G₂₆C₂₅T₂₄ A ₂₄G₂₄C₁₇T₁₇ A ₂₆G₂₈C₂₀T₁₆rickettsii

[0089] The sequence of B. anthracis and B. cereus in region 16S_(—)971is shown below. Shown in bold is the single base difference between thetwo species which can be detected using the methods of the presentinvention. B. anthracis has an ambiguous base at position 20.B.anthracis_16S_971GCGAAGAACCUUACCAGGUNUUGACAUCCUCUGACAACCCUAGAGAUAGGGCUUCUCCUUC (SEQ IDNO: 1) GGGAGCAGAGUGACAGGUGGUGCAUGGUU B.cereus_16S_971GCGAAGAACCUUACCAGGUCUUGACAUCCUCUGAAAACCCUAGAGAUAGGGCUUCUCCUUC (SEQ IDNO: 2) GGGAGCAGAGUGACAGGUGGUGCAUGGUU

EXAMPLE 6 ESI-TOF MS of sspE 56-mer Plus Calibrant

[0090] The mass measurement accuracy that can be obtained using aninternal mass standard in the ESI-MS study of PCR products is shown inFIG. 8. The mass standard was a 20-mer phosphorothioate oligonucleotideadded to a solution containing a 56-mer PCR product from the B.anthracis spore coat protein sspE. The mass of the expected PCR productdistinguishes B. anthracis from other species of Bacillus such as B.thuringiensis and B. cereus.

EXAMPLE 7 B. anthracis ESI-TOF Synthetic 16S_(—)1228 Duplex

[0091] An ESI-TOF MS spectrum was obtained from an aqueous solutioncontaining 5 μM each of synthetic analogs of the expected forward andreverse PCR products from the nucleotide 1228 region of the B. anthracis16S rRNA gene. The results (FIG. 9) show that the molecular weights ofthe forward and reverse strands can be accurately determined and easilydistinguish the two strands. The [M−21H⁺]²¹⁻ and [M−20H⁺]²⁰⁻ chargestates are shown.

EXAMPLE 8 ESI-FTICR-MS of Synthetic B. anthracis 16S_(—)1337 46 BasePair Duplex

[0092] An ESI-FTICR-MS spectrum was obtained from an aqueous solutioncontaining 5 μM each of synthetic analogs of the expected forward andreverse PCR products from the nucleotide 1337 region of the B. anthracis16S rRNA gene. The results (FIG. 10) show that the molecular weights ofthe strands can be distinguished by this method. The [M−16H⁺]¹⁶⁻ through[M−10H⁺]¹⁰⁻ charge states are shown. The insert highlights theresolution that can be realized on the FTICR-MS instrument, which allowsthe charge state of the ion to be determined from the mass differencebetween peaks differing by a single 13C substitution.

EXAMPLE 9 ESI-TOF MS of 56-mer Oligonucleotide from saspB gene of B.anthracis with Internal Mass Standard

[0093] ESI-TOF MS spectra were obtained on a synthetic 56-meroligonucleotide (5 μM) from the saspB gene of B. anthracis containing aninternal mass standard at an ESI of 1.7 μL/min as a function of sampleconsumption. The results (FIG. 11) show that the signal to noise isimproved as more scans are summed, and that the standard and the productare visible after only 100 scans.

EXAMPLE 10 ESI-TOF MS of an Internal Standard with Tributylammonium(TBA)-Trifluoroacetate (TFA) Buffer

[0094] An ESI-TOF-MS spectrum of a 20-mer phosphorothioate mass standardwas obtained following addition of 5 mM TBA-TFA buffer to the solution.This buffer strips charge from the oligonucleotide and shifts the mostabundant charge state from [M−8H⁺]⁸⁻ to [M−3H⁺]³⁻ (FIG. 12).

1 7 1 90 RNA Bacillus anthracis misc_feature (20)..(20) N = A, U, G or C1 gcgaagaacc uuaccaggun uugacauccu cugacaaccc uagagauagg gcuucuccuu 60cgggagcaga gugacaggug gugcaugguu 90 2 90 RNA Bacillus cereus 2gcgaagaacc uuaccagguc uugacauccu cugaaaaccc uagagauagg gcuucuccuu 60cgggagcaga gugacaggug gugcaugguu 90 3 1542 RNA Artificial Sequencemisc_feature 16S rRNA consensus sequence 3 nnnnnnnaga guuugaucnuggcucagnnn gaacgcuggc ggnnngcnun anacaugcaa 60 gucgancgnn nnnnnnnnnnnnnnnnnnnn nnnnnnnnnn agnggcnnac gggugaguaa 120 nncnunnnna nnunccnnnnnnnnnggnan annnnnnnga aannnnnnnu aauaccnnau 180 nnnnnnnnnn nnnnaaagnnnnnnnnnnnn nnnnnnnnnn nnnnnngann nnnnnnngnn 240 nnaunagnun guuggunngguaanggcnna ccaagncnnn gannnnuagc ngnncugaga 300 ggnngnncng ccacanuggnacugaganac ggnccanacu ccuacgggag gcagcagunn 360 ggaaunuunn ncaauggnngnaanncugan nnagcnannc cgcgugnnng anganggnnu 420 nnngnungua aannncununnnnnnngang annnnnnnnn nnnnnnnnnn nnnnnnnnnu 480 gacnnuannn nnnnannaagnnncggcnaa cuncgugcca gcagccgcgg uaauacgnag 540 gnngcnagcg uunnncgganunanugggcg uaaagngnnn gnaggnggnn nnnnnngunn 600 nnngunaaan nnnnnngcunaacnnnnnnn nnncnnnnnn nacnnnnnnn cungagnnnn 660 nnagnggnnn nnngaauunnnnguguagng gugnaauncg naganaunng nangaanacc 720 nnungcgaag gcnnnnnncuggnnnnnnac ugacncunan nnncgaaagc nugggnagcn 780 aacaggauua gauacccugguaguccangc nnuaaacgnu gnnnnnunnn ngnnngnnnn 840 nnnnnnnnnn nnnnnnnnnannnaacgnnn uaannnnncc gccuggggag uacgnncgca 900 agnnunaaac ucaaangaauugacggggnc cngcacaagc ngnggagnau guggnuuaau 960 ucgangnnac gcgnanaaccuuaccnnnnn uugacaunnn nnnnnnnnnn nnganannnn 1020 nnnnnnnnnn nnnnnnnnnnnnnacaggug nugcauggnu gucgucagcu cgugnnguga 1080 gnuguugggu uaagucccgnaacgagcgca acccnnnnnn nnnguuncna ncnnnnnnnn 1140 ngngnacucn nnnnnnacugccnnngnnaa nnnggaggaa ggnggggang acgucaanuc 1200 nucaugnccc uuangnnnngggcuncacac nuncuacaau ggnnnnnaca nngngnngcn 1260 annnngnnan nnnnagcnaancnnnnaaan nnnnucnnag uncggaungn nnncugcaac 1320 ucgnnnncnu gaagnngganucgcuaguaa ucgnnnauca gnangnnncg gugaauacgu 1380 ucncgggncu uguacacaccgcccgucann ncangnnagn nnnnnnnncc nnaagnnnnn 1440 nnnnnnncnn nnnngnnnnnnnnnncnang gnnnnnnnnn nganugggnn naagucguaa 1500 caagguancc nuannngaannugnggnugg aucaccuccu un 1542 4 2904 RNA Artificial Sequencemisc_feature 23S rRNA consensus sequence 4 nnnnaagnnn nnaagngnnnnngguggaug ccunggcnnn nnnagncgan gaaggangnn 60 nnnnncnncn nnanncnnnggnnagnngnn nnnnnncnnn nnanccnnng nunuccgaau 120 ggggnaaccc nnnnnnnnnnnnnnnnnnan nnnnnnnnnn nnnnnnnnnn nnnnnnngnn 180 nacnnnnnga anugaaacaucunaguannn nnaggaanag aaannaannn ngauuncnnn 240 nguagnggcg agcgaannngnannagncnn nnnnnnnnnn nnnnnnnnnn nnnannngaa 300 nnnnnuggna agnnnnnnnnnannngguna nannccngua nnnnaaannn nnnnnnnnnn 360 nnnnnnnnnn aguannncnnnncncgngnn annnngunng aannngnnnn gaccannnnn 420 naagncuaaa uacunnnnnnngaccnauag ngnannagua cngugangga aaggngaaaa 480 gnacccnnnn nangggagugaaanagnncc ugaaaccnnn nncnuanaan nngunnnagn 540 nnnnnnnnnn nnnuganngcgunccuuuug nannaugnnn cngnganuun nnnunnnnng 600 cnagnuuaan nnnnnnnngnagncgnagng aaancgagun nnaanngngc gnnnagunnn 660 nngnnnnaga cncgaancnnngugancuan nnaugnncag gnugaagnnn nnguaanann 720 nnnuggaggn ccgaacnnnnnnnnguugaa aannnnnngg augannugug nnungnggng 780 aaanncnaan cnaacnnngnnauagcuggu ucucnncgaa annnnuuuag gnnnngcnun 840 nnnnnnnnnn nnnnggngguagagcacugn nnnnnnnnng gnnnnnnnnn nnnnuacnna 900 nnnnnnnnaa acuncgaaunccnnnnnnnn nnnnnnnngn agnnanncnn ngngngnuaa 960 nnuncnnngu nnanagggnaacancccaga ncnncnnnua aggncccnaa nnnnnnnnua 1020 aguggnaaan gangugnnnnnncnnanaca nnnaggangu uggcuuagaa gcagccancn 1080 uunaaagann gcguaanagcucacunnucn agnnnnnnng cgcngannau nuancgggnc 1140 uaannnnnnn nccgaannnnnngnnnnnnn nnnnnnnnnn nnnnngguag nngagcgunn 1200 nnnnnnnnnn ngaagnnnnnnngnnannnn nnnuggannn nnnnnnagug ngnaugnngn 1260 naunaguanc gannnnnnnngugananncn nnnncnccgn annncnaagg nuuccnnnnn 1320 nangnunnuc nnnnnngggunagucgnnnc cuaagnngag ncnganangn nuagnngaug 1380 gnnannnggu nnauauuccnnnacnnnnnn nnnnnnnnnn nnnnngacgn nnnnngnnnn 1440 nnnnnnnnnn nnnnggnnnnnnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn 1500 nnnnnnnnnn nnnnnnnnnnnnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn 1560 nnnncnngaa aannnnnnnnnnnnnnnnnn nnnnnnnnnc guaccnnaaa ccgacacagg 1620 ungnnnngnn gagnanncnnaggngnnngn nnnaannnnn nnnaaggaac unngcaaanu 1680 nnnnccguan cuucggnanaaggnnnncnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn 1740 nnnnnnnnng nnnnannnannngnnnnnnn cnacuguuua nnaaaaacac agnncnnugc 1800 naanncgnaa gnnganguauanggnnugac nccugcccng ugcnngaagg uuaanngnnn 1860 nnnnnngnnn nngnnnnnnnnnnnannnaa gcccnnguna acggcggnng uaacuauaac 1920 nnuccuaagg uagcgaaauuccuugucggg uaaguuccga ccngcacgaa nggngnaang 1980 annnnnnnnc ugucucnnnnnnnnncncng ngaanuunna nunnnnguna agaugcnnnn 2040 uncncgcnnn nngacggaaagaccccnngn ancuuuacun nannnunnna nugnnnnnnn 2100 nnnnnnnnug unnagnauaggunggagncn nngannnnnn nncgnnagnn nnnnnggagn 2160 cnnnnnugnn auacnacncunnnnnnnnnn nnnnucuaac nnnnnnnnnn nancnnnnnn 2220 nnngacanug nnngnngggnaguuunacug gggcggunnc cuccnaaann guaacggagg 2280 ngnncnaagg unnncunannnnggnnggnn aucnnnnnnn nagunnaann gnanaagnnn 2340 gcnunacugn nagnnnnacnnnncgagcag nnncgaaagn nggnnnuagu gauccggngg 2400 unnnnnnugg aagngccnucgcucaacgga uaaaagnuac ncnggggaua acaggcunau 2460 nnnncccaag aguncanaucgacggnnnng uuuggcaccu cgaugucggc ucnucncauc 2520 cuggggcugn agnnggucccaagggunngg cuguucgccn nuuaaagngg nacgngagcu 2580 ggguunanaa cgucgugagacaguungguc ccuaucngnn gngngngnnn gannnuugan 2640 nngnnnugnn cnuaguacgagaggaccggn nngnacnnan cncuggugnn ncnguugunn 2700 ngccannngc anngcngnnuagcuannunn ggnnnngaua anngcugaan gcaucuaagn 2760 nngaancnnn cnnnnagannagnnnucncn nnnnnnnnnn nnnnnnnnna gnnncnnnnn 2820 agannannnn gungauaggnnngnnnugna agnnnngnna nnnnunnagn nnacnnnuac 2880 uaaunnnncn nnnnncuunnnnnn 2904 5 13 DNA Artificial Sequence misc_feature Primer 5 cgtggtgaccctt 13 6 14 DNA Artificial Sequence misc_feature Primer 6 cgtcgtcaccgcta 14 7 13 DNA Artificial Sequence misc_feature Primer 7 cgtggtacccctt 13

What is claimed is:
 1. A method of identifying an unknown bioagentcomprising: (a) contacting nucleic acid from said bioagent with at leastone pair of oligonucleotide primers which hybridize to sequences of saidnucleic acid, wherein said sequences flank a variable nucleic acidsequence of the bioagent; (b) amplifying said variable nucleic acidsequence to produce an amplification product; (c) determining themolecular mass of said amplification product; and (d) comparing saidmolecular mass to one or more molecular masses of amplification productsobtained by performing steps (a)-(c) on a plurality of known organisms,wherein a match identifies said unknown bioagent.
 2. The method of claim1, wherein said sequences to which said at least one pair ofoligonucleotide primers hybridize are highly conserved.
 3. The method ofclaim 1, wherein said amplifying step comprises polymerase chainreaction.
 4. The method of claim 1, wherein said amplifying stepcomprises ligase chain reaction or strand displacement amplification. 5.The method of claim 1, wherein said bioagent is a bacterium, virus, cellor spore.
 6. The method of claim 1, wherein said nucleic acid isribosomal RNA.
 7. The method of claim 1, wherein said nucleic acidencodes RNase P or an RNA-dependent RNA polymerase.
 8. The method ofclaim 1, wherein said amplification product is ionized prior tomolecular mass determination.
 9. The method of claim 1, furthercomprising the step of isolating nucleic acid from said bioagent priorto contacting said nucleic acid with said at least one pair ofoligonucleotide primers.
 10. The method of claim 1, further comprisingthe step of performing steps (a)-(d) using a different oligonucleotideprimer pair and comparing the results to one or more molecular massamplification product obtained by performing steps (a)-(c) on adifferent plurality of known organisms from those in step (d).
 11. Themethod of claim 1, wherein said one or more molecular masses arecontained in a database of molecular masses.
 12. The method of claim 1,wherein said amplification product is ionized by electrosprayionization, matrix-assisted laser desorption or fast atom bombardment.13. The method of claim 1, wherein said molecular mass is determined bymass spectrometry.
 14. The method of claim 11, wherein said massspectrometry is selected from the group consisting of Fourier transformion cyclotron resonance mass spectrometry (FT-ICR-MS), ion trap,quadrupole, magnetic sector, time of flight (TOF), Q-TOF and triplequadrupole.
 15. The method of claim 1, further comprising performingstep (b) in the presence of an analog of adenine, thymidine, guanosineor cytidine having a different molecular weight than adenosine,thymidine, guanosine or cytidine.
 16. The method of claim 1, whereinsaid oligonucleotide primer comprises a base analog at positions 1 and 2of each triplet within said primer, wherein said base analog binds withincreased affinity to its complement compared to the native base. 17.The method of claim 16, wherein said primer comprises a universal baseat position 3 of each triplet within said primer.
 18. The method ofclaim 16, wherein said base analog is selected from the group consistingof 2,6-diaminopurine, propyne T, propyne G, phenoxazines and G-clamp.19. The method of claim 16, wherein said universal base is selected fromthe group consisting of inosine, guanidine uridine, 5-nitroindole,3-nitropyrrole, dP, dK, and1-(2-deoxy-β-D-ribofuranosyl)-imidazole-4-carboxamide.
 20. A method ofidentifying an unknown bioagent comprising: contacting nucleic acid fromsaid bioagent with at least one pair of oligonucleotide primers whichhybridize to sequences of said nucleic acid, wherein said sequencesflank a variable nucleic acid sequence; amplifying said variable nucleicacid sequence to produce an amplification product; determining the basecomposition of said amplification product; and comparing said basecomposition to one or more base compositions of amplification productsobtained by performing steps (a)-(c) on a plurality of known organisms,wherein a match identifies said unknown bioagent.
 21. The method ofclaim 20, wherein said sequences to which said at least one pair ofoligonucleotide primers hybridize are highly conserved.
 22. The methodof claim 20, wherein said amplifying step comprises polymerase chainreaction.
 23. The method of claim 20, wherein said amplifying stepcomprises ligase chain reaction or strand displacement amplification.24. The method of claim 20, wherein said bioagent is a bacterium, virus,cell or spore.
 25. The method of claim 20, wherein said nucleic acid isribosomal RNA.
 26. The method of claim 20, wherein said nucleic acidencodes RNase P or an RNA-dependent RNA polymerase.
 27. The method ofclaim 20, wherein said amplification product is ionized prior to basecomposition determination.
 28. The method of claim 20, furthercomprising the step of isolating nucleic acid from said bioagent priorto contacting said nucleic acid with said at least one pair ofoligonucleotide primers.
 29. The method of claim 20, further comprisingthe step of performing steps (a)-(d) using a different oligonucleotideprimer pair and comparing the results to one or more base compositionsof amplification product obtained by performing steps (a)-(c) on adifferent plurality of known organisms from those in step (d).
 30. Themethod of claim 20, wherein said one or more base composition signaturesare contained in a database of base composition signatures.
 31. Themethod of claim 20, wherein said amplification product is ionized byelectrospray ionization, matrix-assisted laser desorption or fast atombombardment.
 32. The method of claim 20, wherein said base compositionsignature is determined by mass spectrometry.
 33. The method of claim32, wherein said mass spectrometry is selected from the group consistingof Fourier transform ion cyclotron resonance mass spectrometry(FT-ICR-MS), ion trap, quadrupole, magnetic sector, time of flight(TOF), q-TOF and triple quadrupole.
 34. The method of claim 20, furthercomprising performing step (b) in the presence of an analog of adenine,thymidine, guanosine or cytidine having a different molecular weightthan adenosine, thymidine, guanosine or cytidine.
 35. The method ofclaim 20, wherein said oligonucleotide primer comprises a base analog atpositions 1 and 2 of each triplet within said primer, wherein said baseanalog binds with increased affinity to its complement compared to thenative base.
 36. The method of claim 35, wherein said primer comprises auniversal base at position 3 of each triplet within said primer.
 37. Themethod of claim 35, wherein said base analog is selected from the groupconsisting of 2,6-diaminopurine, propyne T, propyne G, phenoxazines andG-clamp.
 38. The method of claim 36, wherein said universal base isselected from the group consisting of inosine, guanidine uridine,5-nitroindole, 3-nitropyrrole, dP, dK, and1-(2-deoxy-β-D-ribofuranosyl)-imidazole-4-carboxamide.
 39. A method fordetecting a single nucleotide polymorphism in an individual, comprisingthe steps of: isolating nucleic acid from said individual; contactingsaid nucleic acid with oligonucleotide primers which hybridize toregions of said nucleic acid which flank a region comprising saidpotential polymorphism; amplifying said region to produce anamplification product; determining the molecular mass of saidamplification product; comparing said molecular mass to the molecularmass of said region in an individual known to have said polymorphism,wherein if said molecular masses are the same then said individual hassaid polymorphism.
 40. The method of claim 39, wherein said polymorphismis associated with a disease.
 41. The method of claim 39, wherein saidpolymorphism is a blood group antigen.
 42. The method of claim 39,wherein said amplification step is the polymerase chain reaction. 43.The method of claim 39, wherein said amplification step is ligase chainreaction or strand displacement amplification.
 44. The method of claim39, wherein said amplification product is ionized prior to massdetermination.
 45. The method of claim 39, wherein said amplificationproduct is ionized by electrospray ionization, matrix-assisted laserdesorption or fast atom bombardment.
 46. The method of claim 39, whereinsaid primers hybridize to conserved sequences.
 47. The method of claim39, wherein said molecular mass is determined by mass spectrometry. 48.The method of claim 47, wherein said mass spectrometry is selected fromthe group consisting of Fourier transform ion cyclotron resonance massspectrometry (FT-ICR-MS), ion trap, quadrupole, magnetic sector, time offlight (TOF), Q-TOF and triple quadrupole.